Mast cells (MCs) and T lymphocytes are two cell types integral to development of an allergic response and asthma. The signature response of each of these cells, degranulation and cytokine production, respectively, is induced primarily by cross-linking of the receptor for antigen. In addition, both mast cells and T cells express numerous inflammation-generating receptors coupled to heterotrimeric G proteins (GPCRs). The purpose of this study is to understand mechanisms of intracellular G-protein-coupled signal transduction in these cells and subsequent pathways to inflammation. In particular, the project focuses on the control of G protein activity in inflammatory processes by a novel family of regulators of G protein signaling (RGS proteins), which inhibit function of G alpha subunits by increasing their GTPase activity. G alpha subunits oscillate between GDP- (inactive) and GTP- (active) bound forms based on ligand occupancy of the associated receptor. The GTPase accelerating (GAP) activity of RGS proteins limits the time of interaction of active G-alpha and its effectors, resulting in desensitization of GCPR signaling. Despite a growing body of knowledge concerning the biochemical mechanisms of RGS action, little is known about the physiological role of these proteins in native mammalian systems.[unreadable] [unreadable] RGS13, a GAP for Gi and Gq, was found to be expressed in murine and human mast cells (MCs) and B lymphocytes. RGS13 deficient mice were generated to determine the function of RGS13 in these cell types. A targeting vector was constructed that replaces the RGS13 gene with LacZ, and knockout mice containing the targeted gene were generated by homologous recombination. Positive beta-galactosidase staining was observed in cultured, bone marrow-derived and tissue mast cells from knockout mice. RGS13 expression was also identified by immunohistochemistry in skin mast cells. Allergic (MC-dependent) physiological responses were evaluated in these mice. Surprisingly, both cutaneous and systemic anaphylaxis induced by cross-linking of MC antigen receptors with IgE antibody were markedly increased in RGS13-deficient mice. This abnormality was found to be caused by accentuated IgE-evoked degranulation of RGS13-deficient cultured bone marrow-derived mast cells (BMMCs), and reconstitution of these BMMCs with RGS13 inhibited degranulation. In addition, the GAP activity of RGS13 was not required for this effect. These results have uncovered a new physiological function of RGS13 in mast cells. We hypothesize that abnormalities in RGS13 expression or function may exist in allergic patients or in others with increased mast cell activity. [unreadable] Unexpectedly, RGS13 overexpression in an epithelial cell line inhibited cAMP generation induced by stimulation of a Gs-coupled receptor and by forskolin, a direct activator of adenylyl cyclase. The biochemical basis for this effect was investigated using downstream activators of this signaling pathway. We found that RGS13 acts in the nucleus where it binds the activated (phosphorylated) form of the transcription factor CREB, which is the target of the cAMP pathway. RGS13 inhibited CREB binding to DNA. RGS13-deficient B lymphocytes displayed increased CREB DNA binding and transcription of a CREB target gene, OCA-B. [unreadable] An RGS highly homologous to RGS13, RGS16, is highly expressed in mouse and human activated T lymphocytes. Its function in T cell activation and chemotaxis is being studied by production of conditional knockout mice lacking RGS16 in T lymphocytes. Intracellular flow cytometry confirmed RGS16 expression in unfractionated human and mouse T cells, and RGS16 levels were found to be higher in CD8+ than CD4+ cells. Preliminary data suggest high RGS16 expression in germinal center T lymphocytes isolated from mouse lymphoid tissue. Activation and proliferation induced by T cell receptor stimulation as well as chemokine-mediated migration of RGS16-deficient human T cells are being analyzed using siRNA. Jurkat T cells stably transfected with RGS16 siRNA exhibited increased activation in response to CD3 stimulation, as determined by calcium flux, NFAT activity, and intracellular signaling protein phosphorylation (ERK, ZAP70). The biochemical basis for these abnormalities are currently being investigated.